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Dimerization of a PACAP peptide analogue in DMSO via asparagine and aspartic acid residues
Dimerization of a PACAP Peptide Analogue in DMSO Via Asparagine and Aspartic Acid Residues JOANNE C. SEVERS, WAYNE A. FROLAND Analytics Department, Process Sciences, Bayer Healthcare, 800 Dwight Way, Berkeley, California 94710
Received 8 February 2007; revised 3 May 2007; accepted 1 June 2007 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21116
ABSTRACT: To optimize the stability of a peptide development candidate for the treatment of type II diabetes, formulation studies were initiated in organic solvents and compared to results obtained in aqueous solutions. Stability was assessed by reversed phase liquid chromatography (RPLC) and electrospray ionization mass spectrometry (ESI-MS). Previous studies had shown deamidation and hydrolysis to be the primary mechanisms of degradation in aqueous formulations. Surprisingly, the use of an organic solvent did not decrease the rate of degradation and, as presented here, produced degradation products including dimers. We propose here that deamidation can readily occur in polar anhydrous organic solvents such as DMSO and that the dimer forms through intermolecular nucleophilic attack of an amino acid side chain on a stabilized cyclic imide intermediate. ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:1246–1256, 2008
Keywords: stability; deamidation; dehydration; formulation; mass spectrometry; peptide delivery; peptides; physical characterization; physical stability; HPLC (highperformance/pressure liquid chromatography)
INTRODUCTION The native PACAP is a peptide hormone which is a member of a superfamily of peptide hormones, including vasoactive intestinal peptide, glucagon, growth hormone releasing factor, and secretin.1 By binding to different receptors, PACAP initiates a variety of pharmacological activities, one of which is the stimulation of insulin secretion. Three different PACAP receptor types (R1, R2, and R3) have been reported. Activation of R3 receptors leads to the stimulation of insulin secretion. However, R2 activation stimulates glucose release from the liver and without modification PACAP is not suitable to treat type
Correspondence to: Joanne C. Severs (Telephone: (510) 705 4472; Fax: (510) 705 7769; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 97, 1246–1256 (2008) ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association
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II diabetes, as significant side effects will occur. The modification of native PACAP to produce a peptide capable of mediating the secretion of insulin in a glucose-dependant manner, without the concomitant induction of glucose release, would be highly desirable for the treatment of type II diabetes.2,3 In search of a PACAP-like peptide(s) that can be used safely to treat type II diabetes, a variety of analogues were synthesized and a leading candidate, termed R3P66, was identified (see sequence in Fig. 1). Through selective activation of PACAP R3 receptor, R3P66 should increase cAMP to overcome the glucose resistance of the cell. R3P66, like most unmodified polypeptides exhibits a short half-life in vivo. Use of such a short-lived peptide for therapeutic purposes would require frequent administration, which would be practically and economically prohibitive. To increase the duration of action for this relatively unstable peptide drug, various drug-delivery
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Figure 1. Primary sequence of R3P66. Secondary structure shown is that proposed for the peptide upon interaction with the pancreatic cell. In vitro observation of helical structure by CD was possible in the artificial hydrophobic environment of 50% trifluoroethanol.
and stabilizing approaches have been investigated.3–5 Initial studies of the peptide in aqueous formulations had indicated that deamidation and N-terminal hydrolysis were the primary mechanisms of degradation. LC-MS/MS characterization had shown deamidation to occur during stability studies at both Asn9 and Asn28, with no detectable modification of the glutamines. Conformational isomers were also observed, believed to be due to iso-Asp formation. Since water is directly involved in these degradation pathways, it was generally assumed that the stability of a peptide in an organic solvent would be superior. The stability of peptides in organic solvents has not been well characterized. There are only a few reports on this subject including one that illustrates the kinetics of deamidation in cosolvents of alcohol, dioxane, acetonitrile.6 Others discuss the degradation of polypeptides at aspartyl and asparaginyl residues7 and deamidation in cosolvents of glycerol and a viscosity builder, polyvinyl chloride.8 The current report intends to illustrate how a peptide may be degraded in organic solvents in the absence of water. Surprisingly, the use of an organic formulation did not decrease the rate of degradation and, as presented here, produced interesting results. Degradation products were observed at several critical amino acid residues, including aspartic acid, asparagines, and serine. We propose here a chemical mechanism to explain DOI 10.1002/jps
the polypeptide products formed and observed when the peptide was formulated in DMSO.
EXPERIMENTAL Materials R3P66, a 31 amino acid polypeptide with a monoisotopic mass of 3741.3 Da was synthesized by UCB Bioproducts (Braine-L’Allen, Belgium) using solid state synthesis. Its pI is theoretically calculated to be 10.2. This lyophilized material, on receipt, contained approximately 8% moisture and 7% acetic acid. Organic solvents were used as received, including DMSO (Spectrum, 99.9%).
Methods Peptide solutions at 2 or 300 mg/mL (w/w) were prepared by dissolving the peptide either in organic solvent or aqueous buffers. In the preparation of high concentration stability samples in organic solvents, samples were individually prepared directly in small polypropylene vials. After addition of the required amount of solvents, the sample vials were centrifuged for 5 min at 2000g to spin down peptide particles and to remove the trapped air bubbles. All the sample vials were tightly closed and incubated at 408C. Karl Fischer determination was carried out on samples prior to storage and moisture content JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 3, MARCH 2008
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was shown to be 0.1%. Stability samples were removed periodically and stored briefly at 708C. Samples, along with reference standards, were then diluted to 1 mg/mL in an aqueous environment directly before stability indicating assays. Additionally, as part of a separate study on some vials, the initial peptide was reconstituted in water, dialyzed into 0.1% acetic acid and then immediately lyophilized. The sample was then dissolved at a concentration of 2 mg/mL in DMSO. After storage for 2 weeks at 408C the sample was diluted in water to 1 mg/mL for analysis. Peptide analysis was carrried out employing a Waters’ capillary LC system which was run at 4.8 mL/min. employing a 2% acetic acid/acetonitrile gradient system and a 0.32 150 mm C18 RP column. (Symmetry column, Waters, Milford, MA). This LC system was interfaced with a quadrupole time-of-flight mass spectrometer (Waters) with an ESI source employed in positive ion mode (3500 V) with a cone voltage setting of 35 V, source block temperature of 808C and desolvation temperature of 1508C. Size exclusion chromatography (SEC) was carried out with a TosoHaas TSK G2000SWXL column (5 um particle, 7.8 mm 30 cm) at ambient temperature. The mobile phase used was 0.1% TFA/69.9% 200 mM NaCl in Water/30% ACN running at a flow rate of 0.8 mL/min. The runtime was 20 min and detection wavelength was 214 nm. SEC fractions were prepared for chymotrypsin digestion by removing the solvent fraction in a speedvac and then modifying 100 mL (containing 3 mg dried peptide) of the solution to pH 6.7–7 with
1M Trizma base. Chymotrypsin digestion at 378C was then carried out for 90 min and quenched by freezing. To minimize nonspecific cleavage, the pH was optimized to prevent peptide precipitation and for dimer preservation. Care was taken to achieve identical pH conditions in both monomer and dimer fractions, and replicate samples, as well as an original reference standard, were digested and analyzed each time.
RESULTS It was expected that stability studies on the peptide carried out in organic solvents would show decreased rates of degradation relative to those initially carried out in aqueous conditions, which had shown degradation via deamidation and hydrolysis. However, within a few weeks of the study being initialized, degradation products were observed when analyzed chromatographically. In order to accurately determine the identity of the degradation species, and thus make educated decisions on modifications to the peptide drug molecule and/or formulation, chromatographic peaks were analyzed by LC-ESI-MS. The deconvoluted mass derived from the ESI mass spectrum of native peptide is shown in Figure 2. It can be seen that the mass resolution of the spectrometer used is sufficient to clearly distinguish the natural elemental isotopic distribution of the peptide. The monoisotopic mass of the peptide is shown to be 3741.3 Da, as expected from theoretical calculations. In analysis of each stability sample, a reference standard was always
Figure 2. Deconvoluted mass spectrum of synthetic native R3P66 by ESI-QTOFMS. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 3, MARCH 2008
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run under identical conditions to determine experimental versus stability-induced changes. RPHPLC chromatograms of samples after storage at 408C for 4 weeks are shown in Figure 3B–D, relative to reference standard (Fig. 3A). Stability samples of 2 mg/mL R3P66 in an aqueous solution of pH 4.5 (Fig. 3D) showed two major front running peaks that grew larger with time. In these frontrunning peaks, the N-terminally cleaved species desH-, desHS-, desHSD-R3P66, as well as peptide of (M 18) Da mass and peptide of intact molecular weight (M) were identified by MS. It was believed that this peptide of native mass was due to the formation of an iso-aspartyl residue, which is sufficient to cause separation in the RP chromatogram. This was verified by collecting the peak fraction and analyzing it by Edman sequencing. In this particular case, sequencing stopped at Asp8. As has been shown previously,9,10 Edman sequencing is blocked at iso-aspartyl residues. Samples in aqueous pH 6.5 formulations by comparison did not show any (M 18) Da species but rather deamidation and iso-Asp formation.
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In DMSO, at 300 mg/mL concentration (Fig. 3B), R3P66 resulted in a degradant peak at the end of the gradient that was identified by ESI-MS as the dimer of R3P66, with an intact mass of (2M 18) Da. This dimer was not identified by mass in the aqueous samples. The apparent peak at 31 min in aqueous samples is due to the change in the buffer system, and does not contain dimerized peptide. In DMSO, at 2 mg/ mL concentration, two other peaks were visible (Fig. 3C), namely a front running and a postmain peak. The latter was believed to include a cyclic imide derivative of R3P66 as a molecular weight of (M 18) Da was detected. A small amount of desHS-R3P66 was also detected in this peak. In the front running peak desHSD-R3P66 was detected and peptide of intact mass (likely isoAsp). By 9 weeks, the 300 mg/mL sample also showed low levels of these other two peaks. In a 13-week stability sample at 300 mg/mL, the amount of dimer reached a level of 13%. Additionally, a low amount of trimer was also present in this sample, as detected by MS.
Figure 3. RPLC-UV chromatograms of stability samples in various formulations. DOI 10.1002/jps
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RPLC-MS was also carried out on samples from a secondary study carried out at 2 mg/mL. In this study, the peptide was dialyzed into 0.1% acetic acid, aqueous solution, and then lyophilized. The sample was then dissolved in pure DMSO. Samples at the 0-week time point and 2-week time point were analyzed and compared. The chromatogram of this sample is shown in Figure 4. Peaks observed in the 2-week sample, relative to the 0-week sample, contained native R3P66 (M) and multiple separated species of (M 17) Da, (M 18) Da, (M 19) Da, and (M 1) Da (identified by the monoisotopic mass peak in the spectra). No dimeric species were detected. Theoretically, the majority of acetic acid should have been fully removed during the lyophilization process for this sample. Also the pH and moisture levels in this sample were not well recorded. However, these results are shown to provide further evidence for the theory that we are forming stable cyclic imides in DMSO at both Asp and Asn residues and for the mechanism of dimer formation that we propose in the following discussion. Since the peptide drug candidate was intended to be used at a high concentration, understanding the major degradation mechanism of dimerization
was essential. In order to carry out a detailed characterization study of the nature of the dimer being formed in the DMSO stability samples, SEC was used to carefully separate and collect monomer, dimer, and trimer fractions from the 13-week, 408C DMSO stability sample, Figure 5. LC-MS results obtained on the dimer, Figure 6, showed a very low-level monoisotopic mass peak at (2M 19) Da, but a higher level isotope of (2M 18) Da. There was no 2M species, indicating that the dimer was all covalently linked. Any noncovalent dimers formed could have dissociated during the LC-MS process. A low level of desHSR3P66 dimer was also observed. Purified trimer was predominantly of mass (3M 36) Da. To identify the position of the dimeric linkage, a chymotrypsin digestion of R3P66 was carried out. Theoretically, the enzyme should preferentially cleave at Phe and Tyr, producing four peptide fragments as represented in Table 1. LC-MS results obtained are presented in Table 2 for the chymotryptic digests of both monomer (column 1) and dimer (column 2) fractions from the SEC separation. The digests show that the peptide was cleaved at the three theoretically preferred sites, but also after Leu13. Cleavage after leucine has been reported as the next most favorable
Figure 4. RPLC-UV (210 nm) chromatogram of peptide stability sample, (a) 2-week, 408C, (b) initial. Aqueous peptide dialyzed into 0.1% acetic acid then lyophilized. Sample then dissolved at 2 mg/mL in DMSO. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 3, MARCH 2008
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Figure 5. Size exclusion chromatographic separation of 13-week, 408C DMSO stability sample.
chymotrypsin cleavage site after the aromatic residues. A high percentage of the partial digest peptide T1,2 at mass 1167.5 Da is present, due to incomplete digestion after residue Phe6. LC-MS analysis of both monomer and dimer sample blanks, which were fractions subjected to digestion conditions but without enzyme, showed the presence of a small percentage of des-29, 31 only. The digest of the dimer fraction showed the presence of each of the chymotryptic monomeric peptides, but also multiple additional higher mass
species relative to the monomer digest. These ions were all detected at relatively low levels but with sufficient mass resolution for monoisotopic mass identification. The theoretical assignments, based on mass, for each of these additional species are shown in the table. The masses correlate well with various dimeric peptide species. These results suggest that dimers were all covalently linked and that there were multiple, complex products of dimerization. The fact that dimeric species were not observed in the digest of the monomer proves that dimerization did not
Figure 6. Mass spectrum of dimer fraction of R3P66. DOI 10.1002/jps
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Table 1. Theoretical Chymotrypsin Peptides of R3P66 Tryptic Peptide
T1
T2
T3
T4
Residues HSDAVF TDNY TRLRKQVAAKKY LQSIKNKRY Theoretical monoisotopic mass (Da) 674.3 511.2 1460.9 1148.7
occur due employed.
to
the
experimental
conditions
DISCUSSION In comparison to studies in aqueous solutions in which deamidation, iso-Asp, and cyclic imide formation was observed, in DMSO the peptide primarily degraded via the formation of multimeric species. It is suggested that this latter mechanism is occurring via the cyclic imide intermediate. The formation of the cyclic imide intermediate has been well characterized in peptides. Both deamidation of Asn residues or dehydration of Asp residues can lead to the formation of the intermediate. Opening of the intermediate ring would lead to formation of cleaved products and isomers.
Figure 7 shows the structural changes in these steps. The mechanism and kinetics of secretin degradation via cyclic imide intermediates of Asp3 in aqueous solutions have been reported previously.11 The sites of cyclic imide formation in the peptide under study could be any of the two asparagines (Asn9 or Asn28) or two Aspartic acids (Asp3 or Asp8), leading to (M 17) Da and (M 18) Da products, respectively, as observed in our results. In neutral and weakly alkalinic solution, with comparable sequence, Asn is generally the predominant site of cyclic imide formation.12 At a lower pH, Asp is predominant. Cyclic imide formation has been shown dependent on temperature, pH, neighboring residues, peptide conformation, and polarity of solvent.7 In DMSO, there are no reports showing relative reactivity of Asn or Asp. Given the location of the four residues,
Table 2. LC-QTOF-MS Results for the Chymotrypsin Digests of Monomer and Dimer SEC Fractions of R3P66 Monomer Digest: Monoisotopic Mass (Da) 674.3 701.4 881.46 1148.7 1167.5 1460.9 1537.7 / 1954.1 (minor) / / / / / / / 3741.2
Dimer Digest: Monoisotopic Mass (Da)
Proposed Assignment
Theor. Mass (Da)
674.3 701.4 (minor) 881.5 1148.8 1167.5 (major) 1461 1537.7 1804.0 1954.1 2012.1 2116.2 2222.3 2280.2 2297.2 2298.2 2592.6 (major) 2609.5 3741.2 (minor)
T1 LQSIKN TDNYTRL T4 T1,2 T3 HSDAVFTDNYTRL T1 þ T4 18 1 T2 þ T3 18 or T2,3 TDNYTRL þ T4 18 T1 þ T3 18 1 HSDAVFTDNYTRL þ LQSIKN-17 T4 þ T4 17 T1,2 þ T4 18 1 T1,2 þ T4 18 T3 þ T4 17 T1,2 þ T3 (or T1 þ T2,3) 18 1 Intact
674.3 701.4 881.4 1148.7 1167.5 1460.9 1537.7 1805.0 1 1954.1 2012.2 2117.2 1 2222.1 2280.3 2298.3 1 2298.2 2592.6 2610.5 1 3741.2
Possible Cyclic Imide
D3 D8 D8 D3 N9/N28 N28 D3 D8 N28 D8/D3
Chymotrypsin digestion, 1:10 enzyme: peptide, 90 min, 378C at pH 6.7–7. Asterisks indicate relative intensity. This is only a rough estimation as the ESI signal can be significantly affected by ionization efficiency. Partial digestion peptides are indicated by Tn,n. Dimeric peptides are indicated by Tn þ Tn. Species not observed in digest of monomer. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 3, MARCH 2008
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Figure 7. Reactivity of asparagine residues.
with different adjacent amino acids, it is very difficult to predict which one would be more reactive in DMSO. Mechanistically, cyclic imide formation primarily involves deprotonation of the carboxyl-side backbone amide followed by attack of the nascent anionic nitrogen on the side-chain carbonyl group. Cyclization does not necessarily depend on participation of water but on the presence of a base or hydroxide ion, which initiates the nucleophilic attack on the carbonyl atom. This initial attack leads to the formation of a tetrahedral intermediate, followed by the rate determining loss of amine to give the cyclic imide.13 The cyclic imide intermediate often then undergoes rapid hydrolysis at either carbonyl center in the ring, leading to formation of either Asp or iso-Asp. An alternative, though less favorable cyclic imide pathway is the attack of the side chain amide nitrogen on the peptide bond carbonyl, releasing the carboxyl-flanking peptide, and forming a C-terminal cyclic imide intermediate, see Figure 7.12 The hypothesis postulated from the above results is that the dimerization process contains the following two steps. First, cyclic imide intermediates are forming through deamidation or dehydration at Asn9, Asn28, Asp3, and Asp8. The ring opening reaction is strongly dependent on the presence of a base or hydroxide. Therefore, DOI 10.1002/jps
in aqueous conditions the cyclic intermediate would rapidly open, by direct participation of OH ions even under neutral conditions. In DMSO, however, there is a limit on available nucleophiles in solution. It is proposed that the second step in the dimerization process is nucleophilic attack of a side chain of a second R3P66 peptide, attacking the carbonyl carbon of a stable cyclic imide, causing ring opening and consequently dimer formation. The reaction scheme is represented in Figure 7. The net result is a dimer with 2 R3P66 mass 17 (for Asn) or 18 (for Asp). There are numerous possibilities for such dimers to form, as there are four Asx residues for cyclic imide formation and nine lysine, serine or threonine residues for initiation of a nucleophilic attack. As shown in Table 2, all four Asx residues are implicated in cyclic imide formation. The fact that (M 17) Da and (M 18) Da species are seen suggests that both Asp and Asn residues are involved. Additionally, dimers that contain either no Asp residues (e.g., (T3 or T4) þ T4) or no Asn residues (e.g., T1 þ T3) are observed. Since these peptide species are detected at a low level, the list of peptides in Table 2 may not be exhaustive to account for all the dimers formed. However, there is sufficient evidence from these results to determine that there are indeed multiple dimerization sites. The fact that there is not JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 3, MARCH 2008
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a single predominant reaction route may make it also worth considering whether the oxygen center of the DMSO may be acting as an initial nucleophile opening the cyclic imide ring. The DMSO would then be a highly reactive leaving group, leading to more indiscriminate attack by nucleophilic residues; the amino group of the lysines or the hydroxyl groups of serine and threonine. To add further weight to the hypothesis, lower concentration samples (2 mg/mL) were analyzed, limiting the amount of available nucleophiles to attack the cyclic imide intermediates. It was expected that this could result in sufficiently stable cyclic imides to be detected. This effect was indeed observed as shown in Figure 3C, where species corresponding by mass to both cyclic imides and isomers were observed. The stability samples made up at 300 mg/mL provided a higher concentration of available nucleophiles leading to more dimer formation. A viscosity factor may also be affecting results. Samples that had been previously acidified before formulating in DMSO have subsequently been shown to limit dimerization.5 This appears to also be the case in the acetic acid-treated sample presented in Figure 4. The cyclic imide intermediates (originating from both Asp and Asn) have been stabilized but not attacked by any nucleophiles and have, therefore, not formed dimers. It is surprising, as discussed above, to see the range of dimers produced, as it would be expected that there would be a predominant pathway. It has been established that there is greater structure formation of PACAP analogues in organic solvent. It is believed to have little higher order structure in water alone but forms helixes in the C-terminus in the presence of an organic solvent, as determined by NMR.14 The N-terminal six to eight residues are generally believed disordered. Based on the fact that Asn9 in R3P66 is not in the helix region, we could speculate that it is more vulnerable than the other Asn or Asp residues to formation of cyclic imide. Formation of a cyclic imide intermediate is greatly influenced by the neighboring amino acid on its C-terminal side. Bulky amino acids will reduce the formation of cyclic imide. It has been well established in the literature that Asn with an adjacent glycine is the most reactive. The reported relative rates of cyclic imide formation at pH 7.4 from a model hexapeptide are 6.5, 1.58, and 1 for Asp-Gly, Asp-Ser, and Asp-Ala, respectively. In comparison, Asn-Gly, the most reactive pair, is JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 3, MARCH 2008
232 and Asn-Ala is 13.1.15 This comparison may imply that formation of cyclic imide in Asp is less influenced by the adjacent amino acid residue (6.5-fold) than is the case for Asn (17.1-fold). Detection of cyclic imides derived from Asp residues in protein pharmaceuticals has been reported on several occasions. A cyclic imide residue in place of Asp 129 in porcine somatotropin was observed in acidic solution.16 A succinimide at Asp-130 of methionyl human growth hormone (during storage in sodium phosphate, pH 7.2 at 458C) was also identified by researchers at Genentech.10 Asp is five times faster than Asn in forming succinimide at pH 5, 378C.17 Thus in slight acid environment, the Asp-Ala in R3P66, having a less steric hindered Ala could be more reactive than Asn having bulky adjacent groups such as Asn-Tyr and Asn-Lys. It is believed that this is the first published report supporting the hypothesis of dimerization of a peptide via a stable cyclic imide intermediate. Additionally, cyclic imide formation (or deamidation/dehydration) has not been reported in nonaqueous solutions. Although stable cyclic imides have been reported observed in several cases resulting from Asp residues, a stable cyclic imide has not been reported identified from an Asn residue to our knowledge. Several articles have been published on the dimerization effect in Insulin, which is formed via terminal Asp21 cyclic anhydride intermediate, a more reactive intermediate than the cyclic imide.18,19 A further observation was made from the peptide map data collated in Table 2. It appears that when Asp3 is involved in the dimerization mechanism (and thus T1 is included in the dimer) there is an additional loss of a proton (1 Da). One interpretation is that the free, active N-terminal histidine interacts with a residue sidechain. This phenomenon was also observed in a proportion of the ‘‘degraded’’ intact monomeric peptides under certain conditions. Under these experimental conditions 1 Da from a ‘‘native,’’ monomeric peptide, in either the monomer or dimer digest, was not observed. The N-terminal histidine imidazole base in this class of peptides is known to have a modifying effect.20 It has been reported that N-terminal histidine in secretin caused a specific intramolecular interaction and it plays a crucial role in its biological activity and its chemical properties.20 The increase in pKa’s of histidine in secretin has been suggested to be due to an inductive effect or to a steric effect from remote amino acids. Reports also suggest that it DOI 10.1002/jps
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potentially undergoes intramolecular catalysis with a carboxylic acid group of the Asp320 or interactions with sidechain aromatic residues.21 It has been shown by NMR that there is a difference in the N-terminal His imidazole interaction with Zn in the presence of DMSO relative to aqueous conditions5 and secretin interaction with Zn has been discussed previously.22 It would appear that the loss of an additional proton observed in this case, even if related to the experimental analytical conditions, must also be related to the modified conformation of the peptide in the aged stability samples. As well as degradation via cyclic imides and dimerization, N-terminal degradation of the peptide, in the form of desHS and desHSD (not desH), was also observed when formulated in DMSO, although at a lower level than seen in aqueous conditions.
CONCLUSIONS The use of an organic formulation for PACAPanalogue R3P66 did not decrease the rate of degradation relative to aqueous formulations. The peptide degrades rapidly in organic solvents during incubation at 408C. Analysis of the peptide revealed degradation products, consisting of dimers, polymers, stable cyclic imides, Nterminal hydrolyzed products, and conformational isomers. The major degradation pathway is dimerization. LC-ESI-MS analysis of the digested dimer showed dimerization to have occurred at multiple reaction sites. We propose that dehydration and deamidation can readily occur in polar anhydrous organic solvents such as DMSO and that the dimer forms through intermolecular nucleophilic attack of an amino acid side chain on the cyclic imide intermediate of the deamidation or dehydration degradation reaction. Support for the proposed chemical mechanism was obtained by LC-MS characterization of stability samples of the peptide in DMSO at lower concentrations believed to kinetically limit dimerization. Observation of the cyclic imide intermediate of the proposed mechanism of degradation demonstrates that the chemical mechanism of dehydration and deamidation at aspartic acid and asparagine, respectively, is the same in both polar organic and aqueous systems. Both appear capable of stabilizing transition state structures leading to formation of the cyclic imide intermediate and reaction product. DOI 10.1002/jps
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ACKNOWLEDGMENTS We would like to express our gratitude to the article reviewers for valuable suggestions and comments. Also, to John Wang for helpful discussions and to Melanie Madanat, Wei Wang, and Dennis Chen for Edman sequencing, stability sample preparation, and SEC analysis, respectively.
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10. Teshima G, Stults JT, Ling V, Canova-Davis E. 1991. Isolation and characterization of a succinimide variant of methionyl human growth hormone. J Biol Chem 266:13544–13547. 11. Tsuda T, Uchiyama M, Sato T, Yoshino H, Tsuchiya Y, Ishikawa S, Ohmae M, Watanabe S, Miyake Y. 1990. Mechanism and kinetics of secretin degradation in aqueous solutions. J Pharm Sci 79:223–227. 12. Aswad D. 1995. Deamidation and isoaspartate formation in peptides and proteins Boca Raton: CRC Press. 13. Radkiewicz JL, Zipse H, Clarke S, Houk KN. 2001. Neighboring side chain effects on asparaginyl and aspartyl degradation: An ab initio study of the relationship between peptide conformation and backbone NH acidity. J Am Chem Soc 123:3499– 3506. 14. Wary V, Kakoschke C, Nokihara K, Naruse S. 1993. Solution structure of pituitary adenylate cyclase activating polypeptide by nuclear magnetic resonance spectroscopy. Biochemistry 32:5832–5841. 15. Stephenson RC, Clarke S. 1989. Succinimide formation from aspartyl and asparaginyl peptides as a model for the spontaneous degradation of proteins. J Biol Chem 264:6164–6170. 16. Violand B, Schlittler MR, Kolodziej EW, Toren PC, Cabonce MA, Siegel NR, Duffin KL, Zobel JF, Smith
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21.
22.
CE, Tou JS. 1992. Isolation and characterization of porcine somatotropin containing a succinimide residue in place of aspartate129. Prot Sci 1:1634–1641. Oliyai C, Borchardt RT. 1993. Chemical pathways of peptide degradation IV. Pathways, kinetics and mechanism of degradation of an aspartyl residue in a model hexapeptide. Pharm Res 10:95–102. Darrington RT, Anderson BD. 1995. Effects of insulin concentration and self-association on the partitioning of its A21 cyclic anhydride intermediate to desamido insulin and covalent dimer. Pharm Res 12:1077–1084. Strickley RG, Anderson BD. 1996. Solid-state stability of human insulin I. Mechanism and the effect of water on the kinetics of degradation in lyophiles from pH2-5 solutions. Pharm Res 13: 1142–1152. Hefford MA, Kaplan H. 1989. Chemical properties of the histidine residue of secretin: Evidence for a specific intramolecular interaction. Biochim Biophys Acta 998:267–270. Goossens JF, Cotelle P, Chavatte P, Henichart JP. 1996. NMR study of five N-terminal peptide fragments of the vasoactive intestinal peptide: Crucial role of aromatic residues. Pept Res 9:322–326. Carpenter KA, Schiller PW. 1998. Aggregation behavior and Zn2R binding properties of secretin. Biochemistry 37:16967–16974.
DOI 10.1002/jps
Received 8 February 2007; revised 3 May 2007; accepted 1 June 2007 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21116
ABSTRACT: To optimize the stability of a peptide development candidate for the treatment of type II diabetes, formulation studies were initiated in organic solvents and compared to results obtained in aqueous solutions. Stability was assessed by reversed phase liquid chromatography (RPLC) and electrospray ionization mass spectrometry (ESI-MS). Previous studies had shown deamidation and hydrolysis to be the primary mechanisms of degradation in aqueous formulations. Surprisingly, the use of an organic solvent did not decrease the rate of degradation and, as presented here, produced degradation products including dimers. We propose here that deamidation can readily occur in polar anhydrous organic solvents such as DMSO and that the dimer forms through intermolecular nucleophilic attack of an amino acid side chain on a stabilized cyclic imide intermediate. ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:1246–1256, 2008
Keywords: stability; deamidation; dehydration; formulation; mass spectrometry; peptide delivery; peptides; physical characterization; physical stability; HPLC (highperformance/pressure liquid chromatography)
INTRODUCTION The native PACAP is a peptide hormone which is a member of a superfamily of peptide hormones, including vasoactive intestinal peptide, glucagon, growth hormone releasing factor, and secretin.1 By binding to different receptors, PACAP initiates a variety of pharmacological activities, one of which is the stimulation of insulin secretion. Three different PACAP receptor types (R1, R2, and R3) have been reported. Activation of R3 receptors leads to the stimulation of insulin secretion. However, R2 activation stimulates glucose release from the liver and without modification PACAP is not suitable to treat type
Correspondence to: Joanne C. Severs (Telephone: (510) 705 4472; Fax: (510) 705 7769; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 97, 1246–1256 (2008) ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association
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II diabetes, as significant side effects will occur. The modification of native PACAP to produce a peptide capable of mediating the secretion of insulin in a glucose-dependant manner, without the concomitant induction of glucose release, would be highly desirable for the treatment of type II diabetes.2,3 In search of a PACAP-like peptide(s) that can be used safely to treat type II diabetes, a variety of analogues were synthesized and a leading candidate, termed R3P66, was identified (see sequence in Fig. 1). Through selective activation of PACAP R3 receptor, R3P66 should increase cAMP to overcome the glucose resistance of the cell. R3P66, like most unmodified polypeptides exhibits a short half-life in vivo. Use of such a short-lived peptide for therapeutic purposes would require frequent administration, which would be practically and economically prohibitive. To increase the duration of action for this relatively unstable peptide drug, various drug-delivery
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Figure 1. Primary sequence of R3P66. Secondary structure shown is that proposed for the peptide upon interaction with the pancreatic cell. In vitro observation of helical structure by CD was possible in the artificial hydrophobic environment of 50% trifluoroethanol.
and stabilizing approaches have been investigated.3–5 Initial studies of the peptide in aqueous formulations had indicated that deamidation and N-terminal hydrolysis were the primary mechanisms of degradation. LC-MS/MS characterization had shown deamidation to occur during stability studies at both Asn9 and Asn28, with no detectable modification of the glutamines. Conformational isomers were also observed, believed to be due to iso-Asp formation. Since water is directly involved in these degradation pathways, it was generally assumed that the stability of a peptide in an organic solvent would be superior. The stability of peptides in organic solvents has not been well characterized. There are only a few reports on this subject including one that illustrates the kinetics of deamidation in cosolvents of alcohol, dioxane, acetonitrile.6 Others discuss the degradation of polypeptides at aspartyl and asparaginyl residues7 and deamidation in cosolvents of glycerol and a viscosity builder, polyvinyl chloride.8 The current report intends to illustrate how a peptide may be degraded in organic solvents in the absence of water. Surprisingly, the use of an organic formulation did not decrease the rate of degradation and, as presented here, produced interesting results. Degradation products were observed at several critical amino acid residues, including aspartic acid, asparagines, and serine. We propose here a chemical mechanism to explain DOI 10.1002/jps
the polypeptide products formed and observed when the peptide was formulated in DMSO.
EXPERIMENTAL Materials R3P66, a 31 amino acid polypeptide with a monoisotopic mass of 3741.3 Da was synthesized by UCB Bioproducts (Braine-L’Allen, Belgium) using solid state synthesis. Its pI is theoretically calculated to be 10.2. This lyophilized material, on receipt, contained approximately 8% moisture and 7% acetic acid. Organic solvents were used as received, including DMSO (Spectrum, 99.9%).
Methods Peptide solutions at 2 or 300 mg/mL (w/w) were prepared by dissolving the peptide either in organic solvent or aqueous buffers. In the preparation of high concentration stability samples in organic solvents, samples were individually prepared directly in small polypropylene vials. After addition of the required amount of solvents, the sample vials were centrifuged for 5 min at 2000g to spin down peptide particles and to remove the trapped air bubbles. All the sample vials were tightly closed and incubated at 408C. Karl Fischer determination was carried out on samples prior to storage and moisture content JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 3, MARCH 2008
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was shown to be 0.1%. Stability samples were removed periodically and stored briefly at 708C. Samples, along with reference standards, were then diluted to 1 mg/mL in an aqueous environment directly before stability indicating assays. Additionally, as part of a separate study on some vials, the initial peptide was reconstituted in water, dialyzed into 0.1% acetic acid and then immediately lyophilized. The sample was then dissolved at a concentration of 2 mg/mL in DMSO. After storage for 2 weeks at 408C the sample was diluted in water to 1 mg/mL for analysis. Peptide analysis was carrried out employing a Waters’ capillary LC system which was run at 4.8 mL/min. employing a 2% acetic acid/acetonitrile gradient system and a 0.32 150 mm C18 RP column. (Symmetry column, Waters, Milford, MA). This LC system was interfaced with a quadrupole time-of-flight mass spectrometer (Waters) with an ESI source employed in positive ion mode (3500 V) with a cone voltage setting of 35 V, source block temperature of 808C and desolvation temperature of 1508C. Size exclusion chromatography (SEC) was carried out with a TosoHaas TSK G2000SWXL column (5 um particle, 7.8 mm 30 cm) at ambient temperature. The mobile phase used was 0.1% TFA/69.9% 200 mM NaCl in Water/30% ACN running at a flow rate of 0.8 mL/min. The runtime was 20 min and detection wavelength was 214 nm. SEC fractions were prepared for chymotrypsin digestion by removing the solvent fraction in a speedvac and then modifying 100 mL (containing 3 mg dried peptide) of the solution to pH 6.7–7 with
1M Trizma base. Chymotrypsin digestion at 378C was then carried out for 90 min and quenched by freezing. To minimize nonspecific cleavage, the pH was optimized to prevent peptide precipitation and for dimer preservation. Care was taken to achieve identical pH conditions in both monomer and dimer fractions, and replicate samples, as well as an original reference standard, were digested and analyzed each time.
RESULTS It was expected that stability studies on the peptide carried out in organic solvents would show decreased rates of degradation relative to those initially carried out in aqueous conditions, which had shown degradation via deamidation and hydrolysis. However, within a few weeks of the study being initialized, degradation products were observed when analyzed chromatographically. In order to accurately determine the identity of the degradation species, and thus make educated decisions on modifications to the peptide drug molecule and/or formulation, chromatographic peaks were analyzed by LC-ESI-MS. The deconvoluted mass derived from the ESI mass spectrum of native peptide is shown in Figure 2. It can be seen that the mass resolution of the spectrometer used is sufficient to clearly distinguish the natural elemental isotopic distribution of the peptide. The monoisotopic mass of the peptide is shown to be 3741.3 Da, as expected from theoretical calculations. In analysis of each stability sample, a reference standard was always
Figure 2. Deconvoluted mass spectrum of synthetic native R3P66 by ESI-QTOFMS. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 3, MARCH 2008
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run under identical conditions to determine experimental versus stability-induced changes. RPHPLC chromatograms of samples after storage at 408C for 4 weeks are shown in Figure 3B–D, relative to reference standard (Fig. 3A). Stability samples of 2 mg/mL R3P66 in an aqueous solution of pH 4.5 (Fig. 3D) showed two major front running peaks that grew larger with time. In these frontrunning peaks, the N-terminally cleaved species desH-, desHS-, desHSD-R3P66, as well as peptide of (M 18) Da mass and peptide of intact molecular weight (M) were identified by MS. It was believed that this peptide of native mass was due to the formation of an iso-aspartyl residue, which is sufficient to cause separation in the RP chromatogram. This was verified by collecting the peak fraction and analyzing it by Edman sequencing. In this particular case, sequencing stopped at Asp8. As has been shown previously,9,10 Edman sequencing is blocked at iso-aspartyl residues. Samples in aqueous pH 6.5 formulations by comparison did not show any (M 18) Da species but rather deamidation and iso-Asp formation.
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In DMSO, at 300 mg/mL concentration (Fig. 3B), R3P66 resulted in a degradant peak at the end of the gradient that was identified by ESI-MS as the dimer of R3P66, with an intact mass of (2M 18) Da. This dimer was not identified by mass in the aqueous samples. The apparent peak at 31 min in aqueous samples is due to the change in the buffer system, and does not contain dimerized peptide. In DMSO, at 2 mg/ mL concentration, two other peaks were visible (Fig. 3C), namely a front running and a postmain peak. The latter was believed to include a cyclic imide derivative of R3P66 as a molecular weight of (M 18) Da was detected. A small amount of desHS-R3P66 was also detected in this peak. In the front running peak desHSD-R3P66 was detected and peptide of intact mass (likely isoAsp). By 9 weeks, the 300 mg/mL sample also showed low levels of these other two peaks. In a 13-week stability sample at 300 mg/mL, the amount of dimer reached a level of 13%. Additionally, a low amount of trimer was also present in this sample, as detected by MS.
Figure 3. RPLC-UV chromatograms of stability samples in various formulations. DOI 10.1002/jps
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RPLC-MS was also carried out on samples from a secondary study carried out at 2 mg/mL. In this study, the peptide was dialyzed into 0.1% acetic acid, aqueous solution, and then lyophilized. The sample was then dissolved in pure DMSO. Samples at the 0-week time point and 2-week time point were analyzed and compared. The chromatogram of this sample is shown in Figure 4. Peaks observed in the 2-week sample, relative to the 0-week sample, contained native R3P66 (M) and multiple separated species of (M 17) Da, (M 18) Da, (M 19) Da, and (M 1) Da (identified by the monoisotopic mass peak in the spectra). No dimeric species were detected. Theoretically, the majority of acetic acid should have been fully removed during the lyophilization process for this sample. Also the pH and moisture levels in this sample were not well recorded. However, these results are shown to provide further evidence for the theory that we are forming stable cyclic imides in DMSO at both Asp and Asn residues and for the mechanism of dimer formation that we propose in the following discussion. Since the peptide drug candidate was intended to be used at a high concentration, understanding the major degradation mechanism of dimerization
was essential. In order to carry out a detailed characterization study of the nature of the dimer being formed in the DMSO stability samples, SEC was used to carefully separate and collect monomer, dimer, and trimer fractions from the 13-week, 408C DMSO stability sample, Figure 5. LC-MS results obtained on the dimer, Figure 6, showed a very low-level monoisotopic mass peak at (2M 19) Da, but a higher level isotope of (2M 18) Da. There was no 2M species, indicating that the dimer was all covalently linked. Any noncovalent dimers formed could have dissociated during the LC-MS process. A low level of desHSR3P66 dimer was also observed. Purified trimer was predominantly of mass (3M 36) Da. To identify the position of the dimeric linkage, a chymotrypsin digestion of R3P66 was carried out. Theoretically, the enzyme should preferentially cleave at Phe and Tyr, producing four peptide fragments as represented in Table 1. LC-MS results obtained are presented in Table 2 for the chymotryptic digests of both monomer (column 1) and dimer (column 2) fractions from the SEC separation. The digests show that the peptide was cleaved at the three theoretically preferred sites, but also after Leu13. Cleavage after leucine has been reported as the next most favorable
Figure 4. RPLC-UV (210 nm) chromatogram of peptide stability sample, (a) 2-week, 408C, (b) initial. Aqueous peptide dialyzed into 0.1% acetic acid then lyophilized. Sample then dissolved at 2 mg/mL in DMSO. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 3, MARCH 2008
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Figure 5. Size exclusion chromatographic separation of 13-week, 408C DMSO stability sample.
chymotrypsin cleavage site after the aromatic residues. A high percentage of the partial digest peptide T1,2 at mass 1167.5 Da is present, due to incomplete digestion after residue Phe6. LC-MS analysis of both monomer and dimer sample blanks, which were fractions subjected to digestion conditions but without enzyme, showed the presence of a small percentage of des-29, 31 only. The digest of the dimer fraction showed the presence of each of the chymotryptic monomeric peptides, but also multiple additional higher mass
species relative to the monomer digest. These ions were all detected at relatively low levels but with sufficient mass resolution for monoisotopic mass identification. The theoretical assignments, based on mass, for each of these additional species are shown in the table. The masses correlate well with various dimeric peptide species. These results suggest that dimers were all covalently linked and that there were multiple, complex products of dimerization. The fact that dimeric species were not observed in the digest of the monomer proves that dimerization did not
Figure 6. Mass spectrum of dimer fraction of R3P66. DOI 10.1002/jps
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Table 1. Theoretical Chymotrypsin Peptides of R3P66 Tryptic Peptide
T1
T2
T3
T4
Residues HSDAVF TDNY TRLRKQVAAKKY LQSIKNKRY Theoretical monoisotopic mass (Da) 674.3 511.2 1460.9 1148.7
occur due employed.
to
the
experimental
conditions
DISCUSSION In comparison to studies in aqueous solutions in which deamidation, iso-Asp, and cyclic imide formation was observed, in DMSO the peptide primarily degraded via the formation of multimeric species. It is suggested that this latter mechanism is occurring via the cyclic imide intermediate. The formation of the cyclic imide intermediate has been well characterized in peptides. Both deamidation of Asn residues or dehydration of Asp residues can lead to the formation of the intermediate. Opening of the intermediate ring would lead to formation of cleaved products and isomers.
Figure 7 shows the structural changes in these steps. The mechanism and kinetics of secretin degradation via cyclic imide intermediates of Asp3 in aqueous solutions have been reported previously.11 The sites of cyclic imide formation in the peptide under study could be any of the two asparagines (Asn9 or Asn28) or two Aspartic acids (Asp3 or Asp8), leading to (M 17) Da and (M 18) Da products, respectively, as observed in our results. In neutral and weakly alkalinic solution, with comparable sequence, Asn is generally the predominant site of cyclic imide formation.12 At a lower pH, Asp is predominant. Cyclic imide formation has been shown dependent on temperature, pH, neighboring residues, peptide conformation, and polarity of solvent.7 In DMSO, there are no reports showing relative reactivity of Asn or Asp. Given the location of the four residues,
Table 2. LC-QTOF-MS Results for the Chymotrypsin Digests of Monomer and Dimer SEC Fractions of R3P66 Monomer Digest: Monoisotopic Mass (Da) 674.3 701.4 881.46 1148.7 1167.5 1460.9 1537.7 / 1954.1 (minor) / / / / / / / 3741.2
Dimer Digest: Monoisotopic Mass (Da)
Proposed Assignment
Theor. Mass (Da)
674.3 701.4 (minor) 881.5 1148.8 1167.5 (major) 1461 1537.7 1804.0 1954.1 2012.1 2116.2 2222.3 2280.2 2297.2 2298.2 2592.6 (major) 2609.5 3741.2 (minor)
T1 LQSIKN TDNYTRL T4 T1,2 T3 HSDAVFTDNYTRL T1 þ T4 18 1 T2 þ T3 18 or T2,3 TDNYTRL þ T4 18 T1 þ T3 18 1 HSDAVFTDNYTRL þ LQSIKN-17 T4 þ T4 17 T1,2 þ T4 18 1 T1,2 þ T4 18 T3 þ T4 17 T1,2 þ T3 (or T1 þ T2,3) 18 1 Intact
674.3 701.4 881.4 1148.7 1167.5 1460.9 1537.7 1805.0 1 1954.1 2012.2 2117.2 1 2222.1 2280.3 2298.3 1 2298.2 2592.6 2610.5 1 3741.2
Possible Cyclic Imide
D3 D8 D8 D3 N9/N28 N28 D3 D8 N28 D8/D3
Chymotrypsin digestion, 1:10 enzyme: peptide, 90 min, 378C at pH 6.7–7. Asterisks indicate relative intensity. This is only a rough estimation as the ESI signal can be significantly affected by ionization efficiency. Partial digestion peptides are indicated by Tn,n. Dimeric peptides are indicated by Tn þ Tn. Species not observed in digest of monomer. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 3, MARCH 2008
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Figure 7. Reactivity of asparagine residues.
with different adjacent amino acids, it is very difficult to predict which one would be more reactive in DMSO. Mechanistically, cyclic imide formation primarily involves deprotonation of the carboxyl-side backbone amide followed by attack of the nascent anionic nitrogen on the side-chain carbonyl group. Cyclization does not necessarily depend on participation of water but on the presence of a base or hydroxide ion, which initiates the nucleophilic attack on the carbonyl atom. This initial attack leads to the formation of a tetrahedral intermediate, followed by the rate determining loss of amine to give the cyclic imide.13 The cyclic imide intermediate often then undergoes rapid hydrolysis at either carbonyl center in the ring, leading to formation of either Asp or iso-Asp. An alternative, though less favorable cyclic imide pathway is the attack of the side chain amide nitrogen on the peptide bond carbonyl, releasing the carboxyl-flanking peptide, and forming a C-terminal cyclic imide intermediate, see Figure 7.12 The hypothesis postulated from the above results is that the dimerization process contains the following two steps. First, cyclic imide intermediates are forming through deamidation or dehydration at Asn9, Asn28, Asp3, and Asp8. The ring opening reaction is strongly dependent on the presence of a base or hydroxide. Therefore, DOI 10.1002/jps
in aqueous conditions the cyclic intermediate would rapidly open, by direct participation of OH ions even under neutral conditions. In DMSO, however, there is a limit on available nucleophiles in solution. It is proposed that the second step in the dimerization process is nucleophilic attack of a side chain of a second R3P66 peptide, attacking the carbonyl carbon of a stable cyclic imide, causing ring opening and consequently dimer formation. The reaction scheme is represented in Figure 7. The net result is a dimer with 2 R3P66 mass 17 (for Asn) or 18 (for Asp). There are numerous possibilities for such dimers to form, as there are four Asx residues for cyclic imide formation and nine lysine, serine or threonine residues for initiation of a nucleophilic attack. As shown in Table 2, all four Asx residues are implicated in cyclic imide formation. The fact that (M 17) Da and (M 18) Da species are seen suggests that both Asp and Asn residues are involved. Additionally, dimers that contain either no Asp residues (e.g., (T3 or T4) þ T4) or no Asn residues (e.g., T1 þ T3) are observed. Since these peptide species are detected at a low level, the list of peptides in Table 2 may not be exhaustive to account for all the dimers formed. However, there is sufficient evidence from these results to determine that there are indeed multiple dimerization sites. The fact that there is not JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 3, MARCH 2008
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a single predominant reaction route may make it also worth considering whether the oxygen center of the DMSO may be acting as an initial nucleophile opening the cyclic imide ring. The DMSO would then be a highly reactive leaving group, leading to more indiscriminate attack by nucleophilic residues; the amino group of the lysines or the hydroxyl groups of serine and threonine. To add further weight to the hypothesis, lower concentration samples (2 mg/mL) were analyzed, limiting the amount of available nucleophiles to attack the cyclic imide intermediates. It was expected that this could result in sufficiently stable cyclic imides to be detected. This effect was indeed observed as shown in Figure 3C, where species corresponding by mass to both cyclic imides and isomers were observed. The stability samples made up at 300 mg/mL provided a higher concentration of available nucleophiles leading to more dimer formation. A viscosity factor may also be affecting results. Samples that had been previously acidified before formulating in DMSO have subsequently been shown to limit dimerization.5 This appears to also be the case in the acetic acid-treated sample presented in Figure 4. The cyclic imide intermediates (originating from both Asp and Asn) have been stabilized but not attacked by any nucleophiles and have, therefore, not formed dimers. It is surprising, as discussed above, to see the range of dimers produced, as it would be expected that there would be a predominant pathway. It has been established that there is greater structure formation of PACAP analogues in organic solvent. It is believed to have little higher order structure in water alone but forms helixes in the C-terminus in the presence of an organic solvent, as determined by NMR.14 The N-terminal six to eight residues are generally believed disordered. Based on the fact that Asn9 in R3P66 is not in the helix region, we could speculate that it is more vulnerable than the other Asn or Asp residues to formation of cyclic imide. Formation of a cyclic imide intermediate is greatly influenced by the neighboring amino acid on its C-terminal side. Bulky amino acids will reduce the formation of cyclic imide. It has been well established in the literature that Asn with an adjacent glycine is the most reactive. The reported relative rates of cyclic imide formation at pH 7.4 from a model hexapeptide are 6.5, 1.58, and 1 for Asp-Gly, Asp-Ser, and Asp-Ala, respectively. In comparison, Asn-Gly, the most reactive pair, is JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 3, MARCH 2008
232 and Asn-Ala is 13.1.15 This comparison may imply that formation of cyclic imide in Asp is less influenced by the adjacent amino acid residue (6.5-fold) than is the case for Asn (17.1-fold). Detection of cyclic imides derived from Asp residues in protein pharmaceuticals has been reported on several occasions. A cyclic imide residue in place of Asp 129 in porcine somatotropin was observed in acidic solution.16 A succinimide at Asp-130 of methionyl human growth hormone (during storage in sodium phosphate, pH 7.2 at 458C) was also identified by researchers at Genentech.10 Asp is five times faster than Asn in forming succinimide at pH 5, 378C.17 Thus in slight acid environment, the Asp-Ala in R3P66, having a less steric hindered Ala could be more reactive than Asn having bulky adjacent groups such as Asn-Tyr and Asn-Lys. It is believed that this is the first published report supporting the hypothesis of dimerization of a peptide via a stable cyclic imide intermediate. Additionally, cyclic imide formation (or deamidation/dehydration) has not been reported in nonaqueous solutions. Although stable cyclic imides have been reported observed in several cases resulting from Asp residues, a stable cyclic imide has not been reported identified from an Asn residue to our knowledge. Several articles have been published on the dimerization effect in Insulin, which is formed via terminal Asp21 cyclic anhydride intermediate, a more reactive intermediate than the cyclic imide.18,19 A further observation was made from the peptide map data collated in Table 2. It appears that when Asp3 is involved in the dimerization mechanism (and thus T1 is included in the dimer) there is an additional loss of a proton (1 Da). One interpretation is that the free, active N-terminal histidine interacts with a residue sidechain. This phenomenon was also observed in a proportion of the ‘‘degraded’’ intact monomeric peptides under certain conditions. Under these experimental conditions 1 Da from a ‘‘native,’’ monomeric peptide, in either the monomer or dimer digest, was not observed. The N-terminal histidine imidazole base in this class of peptides is known to have a modifying effect.20 It has been reported that N-terminal histidine in secretin caused a specific intramolecular interaction and it plays a crucial role in its biological activity and its chemical properties.20 The increase in pKa’s of histidine in secretin has been suggested to be due to an inductive effect or to a steric effect from remote amino acids. Reports also suggest that it DOI 10.1002/jps
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potentially undergoes intramolecular catalysis with a carboxylic acid group of the Asp320 or interactions with sidechain aromatic residues.21 It has been shown by NMR that there is a difference in the N-terminal His imidazole interaction with Zn in the presence of DMSO relative to aqueous conditions5 and secretin interaction with Zn has been discussed previously.22 It would appear that the loss of an additional proton observed in this case, even if related to the experimental analytical conditions, must also be related to the modified conformation of the peptide in the aged stability samples. As well as degradation via cyclic imides and dimerization, N-terminal degradation of the peptide, in the form of desHS and desHSD (not desH), was also observed when formulated in DMSO, although at a lower level than seen in aqueous conditions.
CONCLUSIONS The use of an organic formulation for PACAPanalogue R3P66 did not decrease the rate of degradation relative to aqueous formulations. The peptide degrades rapidly in organic solvents during incubation at 408C. Analysis of the peptide revealed degradation products, consisting of dimers, polymers, stable cyclic imides, Nterminal hydrolyzed products, and conformational isomers. The major degradation pathway is dimerization. LC-ESI-MS analysis of the digested dimer showed dimerization to have occurred at multiple reaction sites. We propose that dehydration and deamidation can readily occur in polar anhydrous organic solvents such as DMSO and that the dimer forms through intermolecular nucleophilic attack of an amino acid side chain on the cyclic imide intermediate of the deamidation or dehydration degradation reaction. Support for the proposed chemical mechanism was obtained by LC-MS characterization of stability samples of the peptide in DMSO at lower concentrations believed to kinetically limit dimerization. Observation of the cyclic imide intermediate of the proposed mechanism of degradation demonstrates that the chemical mechanism of dehydration and deamidation at aspartic acid and asparagine, respectively, is the same in both polar organic and aqueous systems. Both appear capable of stabilizing transition state structures leading to formation of the cyclic imide intermediate and reaction product. DOI 10.1002/jps
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ACKNOWLEDGMENTS We would like to express our gratitude to the article reviewers for valuable suggestions and comments. Also, to John Wang for helpful discussions and to Melanie Madanat, Wei Wang, and Dennis Chen for Edman sequencing, stability sample preparation, and SEC analysis, respectively.
REFERENCES 1. Vaudry D, Gonzalez BJ, Basille M, Yon L, Fournier A, Vaudry H. 2000. Pituitary adenylate cyclaseactivating polypeptide and its receptors: From structure to functions. Pharmacol Rev 52:269– 324. 2. Tsutsumi M, Claus TH, Liang Y, Li Y, Yang L, Zhu J, Dela Cruz F, Peng X, Chen H, Yung SL, Hamren S, Livingston JN, Pan CQ. 2002. A potent and highly selective VPAC2 agonist enhances glucoseinduced insulin release and glucose disposal A potential therapy for type 2 diabetes. Diabetes 51: 1453–1460. 3. Yung SL, Cruz FD, Hamren S, Zhu J, Tsutsumi M, Bloom JW, Caudle M, Roczniak S, Todd T, Lemoine L, MacDougall M, Shanafelt AB, Pan CQ. 2003. Generation of highly selective VPAC2 receptor agonists by high throughput mutagenesis of vasoactive intestinal peptide and pituitary adenylate cyclase-activating peptide. J Biol Chem 278: 10273–10281. 4. Pan C, Li F, Tom I, Wang W, Dumas M, Froland W, Yung SL, Li Y, Roczniak S, Claus TH, Wang YJ, Whelan JP. 2007. Engineering novel VPAC2 selective agonists with improved stability and glucose lowering activity in vivo. J Pharmacol Exp Ther 330:900–906. 5. Wang W, Martin-Moe S, Pan C, Musza L, Wang JY. 2007. Stabilization of a polypeptide in nonaqueous solvents. Int J Pharm (submitted for publication). 6. Capasso S, Mazzarella L, Zagari A. 1991. Deamidation via cyclic imide of asparaginyl peptides: Dependence on salts, buffers and organic solvents. Pept Res 4:234–238. 7. Brennan TV, Clark S. 1993. Spontaneous degradation of polypeptides at aspartyl and asparaginyl residues: Effects of the solvent dielectric. Prot Sci 2:331–338. 8. Li R, D’Souza AJ, Laird BB, Schowen RL, Borchardt RT, Topp EM. 2000. Effects of solution polarity and viscosity on peptide deamidation. J Pept Res 56: 326–334. 9. Smyth DG, Stein WH, Moore S. 1962. On the sequence of residues 11 to 18 in bovine pancreatic ribonuclease. J Biol Chem 237:1845–1850. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 3, MARCH 2008
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DOI 10.1002/jps